Neurons are selectively venerable with a low stressor-threshold in neurodegenerative diseases. At molecular levels, responses to cellular stresses are mediated by dynamic protein-protein interactions (PPI). Developing in-depth, dynamic PPI networks is therefore crucial to understand the pathogenesis of these diseases. Huntington?s disease (HD) is an inherited fatal neurodegenerative disorder caused by a mutation in the huntingtin (htt) gene. It is strongly suggested that Htt serves as a scaffold protein interacting with multiple protein complexes that are involved in diverse cellular functions. Our long-range goal is to understand the molecular functions of Htt and muHtt at various biological states. The objective of this multi- PI proposal is to map Htt/muHtt interactome under the proteotoxic stress. Our central hypothesis is that normal Htt remodels its interactome in response to cellular stresses and this capability is compromised in the presence of muHtt, causing accumulation of cellular damages overtime and eventually neurodegeneration. Specifically, the following two aims are proposed. Aim 1: Map the dynamic Htt/muHtt interactome in response to proteotoxic stress by unbiased quantitative proteomic and bioinformatic analyses. We will first establish an ascorbate peroxidase (APEX2)-based proximity labeling platform to spatiotemporally label Htt-interacting proteins in live cells. A striatal STHdhQ7 neuronal cell line stably expressing Htt-APEX2 will be subjected to three different conditions (normal, proteotoxic stress and stress recovery) followed by APEX2 labeling. Biotinylated proteins will then be identified by quantitative proteomics. The resulting protein list will be subjected to in-depth bioinformatic analyses. Aim 2: Quantitatively analyze the molecular responses of known Htt-interacting proteins to cellular stresses in normal and HD cells. We will focus on analyzing a signaling hub protein, p62, which directly interacts with Htt. Our working hypothesis is that Htt regulates the molecular responses of p62 to cellular stresses and the regulation is impaired in the presence of muHtt. To test this hypothesis, molecular changes of p62 to various stresses in normal and HD cells will be evaluated at (1) mRNA levels, (2) protein expression and (3) subcellular localization. The interaction between p62 and Htt/muHtt under various stresses will be quantified using the Htt-APEX2 platform. We are well-positioned to undertake the proposed study because our research team consists of uniquely qualified individuals with combined expertise in molecular neurobiology and large data analysis. Successful completion of these studies will contribute fundamental knowledge about molecular functions of normal and mutant Htt and the pathogenesis of HD. In a broader aspect, building dynamic interaction networks under diverse stress conditions could be the key to understand the molecular differences between healthy and any pathological states. The proposed research is highly innovative for its novel idea and approaches to study the dynamic nature of Htt interactome in response to stresses.